T Cell Receptor Assembly
T-cells are a subgroup of cells which together with other immune cell types (polymorphonuclear, eosinophils, basophils, mast cells, B-, NK cells), constitute the cellular component of the immune system. Under physiological conditions T-cells function in immune surveillance and in the elimination of foreign antigen. However, under pathological conditions there is compelling evidence that T-cells play a major role in the causation and propagation of disease. In these disorders, breakdown of T-cell immunological tolerance, either central or peripheral is a fundamental process in the causation of autoimmune disease.
Central tolerance involves thymic deletion of self reactive cells (negative selection) and positive selection of T-cells with low affinity for self major histocompatibility complex antigens (MHC). In contrast, there are four, non-mutually exclusive hypotheses that have been proposed to explain peripheral T-cell tolerance which are involved in the prevention of tissue specific autoimmune disease. These include: anergy (loss of co-stimulatory signals, down regulation of receptors critical for T-cell activation), deletion of reactive T-cells, ignorance of the antigen by the immune system and suppression of autoreactive T-cells. Tolerance once induced does not necessarily persist indefinitely. A breakdown in any of these mechanisms may lead to auto-immune disease.
Autoimmune disease and other T-cell mediated disorders are characterised by the recruitment of T-cells to sites of inflammation. T-cells at these sites, coupled with their ability to produce and regulate cytokines and influence B-cell function, orchestrate the immune response and shape the final clinical outcome. An understanding of the process of T-cell antigen recognition and subsequent T-cell activation, leading to T-cell proliferation and differentiation, is therefore pivotal to both health and disease. Disturbance in this intricate structure-function relationship of the T-cell antigen receptor, harmonising antigen recognition with T-cell activation may provide the therapeutic means to deal with inflammation and T-cell mediated disorders.
The TCR is composed of at least seven transmembrane proteins1. The disulfide-linked (αβ-Ti) heterodimer forms the monotypic antigen recognition unit, while the invariant chains of CD3, consisting of ε, γ, δ, and ζ and η chains, are responsible for coupling the ligand binding to signalling pathways that result in T-cell activation and the elaboration of the cellular immune responses. Despite the gene diversity of the TCR chains, two structural features are common to all known subunits. Firstly, they are transmembrane proteins with a single transmembrane spanning domain—presumably alpha-helical. Secondly, all the TCR chains have the unusual feature of possessing a charged amino acid within the predicted transmembrane domain. The invariant chains have a single negative charge, conserved between the mouse and human, and the variant chains possess one (TCR-β) or two (TCR-α) positive charges. Listed in Table 1 is the transmembrane sequence of TCR-α in a number of species showing that this region is highly conserved and that phylogenetically may subserve an important functional role. The octapeptide (bold) containing the hydrophilic amino acids arginine and lysine is identical between the species. The amino acid substitutions noted in the remaining portions of the transmembrane sequence are minor and conservative.
TABLE 1Sequence comparison of TCR-α transmembraneregion in several speciesSPECIESSEQUENCEMOUSENLSVMGLRILLLKVAGFNLLMTLSEQ ID NO. 1RATNLSVMGLRILLLKVAGFNLLMTLSEQ ID NO. 2SHEEPNLSVTVFRILLLKVVGFNLLMTLSEQ ID NO. 3COWNLSVI VFRILLLKVVGFNLLMTLSEQ ID NO. 4HUMANNLSVI GFRILLLKVAGFNLLMTLSEQ ID NO. 5Studies on the assembly of the multicomponent TCR by Manolios et al2,3,4 showed that the stable interaction between TCR-α and CD3-δ and TCR-α and CD3-ε was localised to eight amino acids within the transmembrane domain of TCR-α and it was the charged amino acids arginine and lysine that were critical for this process. This finding exemplified the fact that amino acids within the transmembrane domain not only functioned to anchor proteins but were important in the assembly of subunit complexes and protein—protein interactions. For the first time it was found that the assembly of this complex receptor could hinge on only eight amino acids. The above system depended on the modification of complementary strand DNA (cDNA) to create a number of protein mutants. Chimeric cDNA molecules were transfected into COS cells to express the required protein. Coexpression of these chimeric proteins were used to evaluate the region of interaction. The technology involved cDNA manipulation, metabolic labelling, immunoprecipitation and gel electrophoresis. Transmembrane domains are small in size and proteins transversing this region are constrained to an alpha-helical configuration. These biophysical features coupled with the ability to engineer protein—protein interactions via transmembrane charge groups suggested a possible new approach to intervene and potentially disturb TCR function. The use of peptides as possible inhibitors of assembly, the recognition and application of this peptide sequence as a possible therapeutic agent to interfere with T-cell function was not a normal or obvious extension.
In co-pending International Patent Application No. PCT/AU96/00018 the present inventor developed peptides which disturb TCR function. The disclosure of this application is included herein by cross-reference5.
Biologics in the Treatment of Inflammatory Disease.
In the last decade a new age of therapeutics has developed with the so-called “Biologics”, that aim to target specific individual cells, and molecules within the cells, with the specific purpose of interrupting immunological networks and cascades thought to underlie the disease process. The disease model for rheumatoid arthritis has been exemplary in the design of biological agents and a number of different approaches have been devised and tested6. The model predicts that an initial arthritogenic peptide is presented to T-cells by an antigen presenting cell (APC) which causes activation of T cells and release of cytokines and proteases culminating in chronic inflammation and joint damage (FIG. 1a). Based on this model a large number of different potentially therapeutic strategies have been devised and used to interfere with the interaction between TCR, MHC and antigen (trimolecular complex) and thereby influence the immune response. Early therapeutic attempts at reducing circulating lymphocyte numbers, included nodal irradiation7, thoracic duct drainage8 and lymphocytapheresis9. Newer sites of lymphocyte intervention are numbered (1–5) in FIG. 1a and include the use of monoclonal antibodies (MAbs) to either delete T-cells or regulate their function, T-cell vaccines against the pathogenic T-cells, the synthesis of analogous peptides to compete with the antigenic peptide, and inhibition of cytokine action following T-cell activation. These new immunomodulatory therapeutic approaches have been applied in animal models, of spontaneously or experimentally induced autoimmune disease, with encouraging results. These approaches are now being used in human autoimmune diseased. More novel approaches focus on eliminating or modulating T-cells by interfering with the delicate trimolecular complex between antigen, T-cell and MHC molecules. Since antigen is recognised by B and/or T cells and subsequent events are based on this interaction, we have reasoned that interfering with the early antigen recognition events (trimolecular complex) may have profound effects on the development of disease, irrespective of what downstream cellular and cytokine events may occur.
The trimolecular complex as the site for therapeutic intervention has been the subject of focus since the recent advances in the molecular characterisation of its constituents and has provided several approaches for immune intervention. The aim of therapy is to eliminate, prevent or downregulate the T-cell response by a variety of means (FIG. 1b).
(i) MAbs to T-cell antigens. The use of MAbs in the treatment of RA has been reviewed by a number of authors6, 10, 11. The MAbs tested were directed against a variety of antigens ranging from: (a) those present on all mature T-cells, and thought to be involved in the pathogenesis of RA (CD5, CDw52)12, 13; (b) MAbs specific for T-cell subsets (CD4), which have the advantage of limited immunosuppressive effects14,15; and (c) to MAbs directed against T-cell activation antigens (IL-2 receptor) which may specifically suppress activated T-cells in response to antigen16,17. All the MAbs used are derived from rodents and only CAMPATH-1H has been “humanised” by recombinant cDNA techniques. Clinical studies indicate that these MAbs are well tolerated in patients and can induce a favourable clinical response. Side effects include an immune reaction to the rodent antibodies which may restrict recurrent use.
(ii) Anti-MHC therapy. Immunogenetic studies have demonstrated that the MHC molecules (DR1, DR4, Dw4 and DR4 Dw14) are important in RA susceptibility18. Since MHC molecules present antigenic peptides to T-cells they provide another target for immune intervention. The function of these molecules can be interfered with either by using MAbs (to the antigen binding sites)19 or high affinity binding of competitor peptides to the MHC groove (see below). MAbs directed against MHC molecules interfere with disease initiation in several animal models of autoimmunity20,21 and humans22.
(iii) Peptide competition. T-cell recognition of antigen can be disrupted by using high affinity MHC-binding peptides which block the antigen-binding site of MHC molecules and inhibit T-cell responses. By substitution of particular amino acid residues it is possible to generate “designer’ peptides, which have high affinity for MHC molecules but do not activate T-cells23. This therapy has the advantage of specificity without causing generalised immunosuppression.
(iv) T-cell vaccination. This form of therapy holds promise for those diseases which exhibit T-cell oligoclonality. The idea is to obtain pathogenic T-cell clones and vaccinate against these cells hoping to eliminate them from the available T-cell repertoire. Another more refined method of vaccination has been to synthesize peptides corresponding to the T-cell receptor sequences which are involved in antigen recognition. Autoimmune animal models vaccinated with such peptides support the view that it is possible to block functional T-cell clones by using synthetic peptides24,25. Whether these antiTCR strategies are applicable to rheumatoid disease depends on the oligoclonality of the autoreactive cells and their limited TCR usage. Although still controversial, evidence of a limited repertoire of TCR usage has been reported in RA26,27.
(v) Cytokine therapy. Synovial fluid analysis of patients with RA has shown the presence of a large number of cytokines including granulocyte-macrophage colony stimulating factor (GM-CSF), gamma-interferon (IFN-γ), interleukin-1 (IL-1) and tumour necrosis factor (TNF-α)28. Cytokines interact with cells to co-ordinate the immune and inflammatory response. They can be grouped as either pro-inflammatory or anti-inflammatory. IL-1 and TNFα are in the former group and act synergistically. TNF-α is also one of the major cytokines regulating the expression of IL-128. Because of their central importance attempts to interfere with their regulation or production may have a positive effect on disease outcome29, 30. Administration of IL-1 receptor antagonist to rats and mice with arthritis has reduced the severity of joint lesions and is in Phase II studies in human disease. Therapeutic use of MAbs to the IL-2 receptor has transient effects31. The receptors for a large group of cytokines have been cloned and sequenced (reviewed by Dower and Sims)32 and currently under clinical evaluation33. It may be that the soluble form of the cytokine receptors may be used to sequester the cytokines by a ligand type interaction and thereby reduce inflammation. Cyclosporin A modulates T-cell cytokine production and when given in several trials has given good clinical response. However the associated nephrotoxicity limits its use34.
(vi) The ability to disrupt cellular function by the use of peptides derived from protein sequences critical for receptor assembly, has only recently been published35 and is a new approach for the use of biologics, that could be included into the schema of biological mechanisms of action. That is, the disruption of cellular function by “disorganising” the assembly of receptors by use of peptides. By design, the peptide chosen corresponded to a common transmembrane sequence common to both CD4 and CD8 cells and currently other unique sites of TCR chain interaction are under investigation. In particular, interactions in the extra cellular domain between the antigen recognition chains, may prove useful in devising peptides for individual pathogenic T cell clones with specific Vα/Vβ usage.